Lentiviral Barcode Labeling and Transplantation of Fetal Liver Hematopoietic Stem and Progenitor Cells
胎儿肝脏造血干细胞和祖细胞的慢病毒条形码标记和移植
发布: 2017年04月20日第7卷第8期 DOI: 10.21769/BioProtoc.2242 浏览次数: 9297
评审: Ivan ZanoniAnupam JhingranAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2016
Abstract
Cellular barcoding enables the dissection of clonal dynamics in heterogeneous cell populations through single cell lineage tracing. The labeling of hematopoietic stem and progenitor cells (HSPCs) with unique and heritable DNA barcodes, makes it possible to resolve donor cell heterogeneity in terms of differentiation potential and lineage bias at the single cell level, through subsequent transplantation and high-throughput sequencing. Furthermore, cellular barcoding allows for bona fide hematopoietic stem cells (HSCs) to be defined based on functional rather than immunophenotypic parameters.
This protocol describes the work flow of lentiviral cellular barcoding, tracking 14.5 days post coitum (d.p.c.) fetal liver (FL) Lineage-Sca+cKit+ (LSK) HSPCs following long-term reconstitution (Figure 1) (Kristiansen et al., 2016), but can be adapted to the cell type or time frame of choice.
Figure 1. Summary of experimental workflow (Naik et al., 2013)
Lentiviral transduction (慢病毒转导)
Fetal liver (胎儿肝脏)
Hematopoietic stem and progenitor cells (造血干细胞和祖细胞)
Single cell lineage tracing (单细胞谱系追踪)
Transplantation (移植)
Background
The cellular barcoding technique was initially established to resolve single cell dynamics upon transplantation of hematopoietic cells in vivo and has in recent years contributed significantly to our appreciation of the functional heterogeneity within blood cell populations in a transplantation setting (Schepers et al., 2008; Gerrits et al., 2010; Lu et al., 2011; Naik et al., 2013; Verovskaya et al., 2013; Kristiansen et al., 2016). The generation and characterization of lentiviral barcode libraries, the importance of library complexity as well as the associated analytical challenges have been carefully reviewed (Bystrykh et al., 2014; Naik et al., 2014; Bystrykh and Belderbosv, 2016) and need to be considered before starting this protocol to ensure proper experimental design. The current protocol pertains our adaptation of the technology as seen in our recent article (Kristiansen et al., 2016), to trace the long-term reconstitution capacity of FL derived HSPCs.
Materials and Reagents
- 96-well non-treated plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 260836 )
- Tissue paper (KCWW, Kimberly-Clark, catalog number: 75512 )
- Autoclaved 1.5 ml Eppendorf tubes (Fisher Scientific, FisherbrandTM, catalog number: 05-408-129 )
- 50 ml tubes (Corning, Falcon®, catalog number: 352070 )
- 1 ml pipette tips (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2179-05-HR )
- Cell strainers, 40 µm (Corning, catalog number: 431750 )
- 60 mm Petri-dishes (Corning, catalog number: 430166 )
- Cup filters, 50 µm (BD, BD Biosciences, catalog number: 340630 )
- 1 ml syringe with 29 gauge ½ inch needle (Terumo Medical, catalog number: BS-N1H2913 )
- FACS tubes (Corning, Falcon®, catalog number: 352058 )
- Mice: Males and females of appropriate experimental genotypes for timed pregnancies
Recipient mice of the appropriate experimental genotype
One mouse of the appropriate experimental genotype for support bone marrow cells
Note: This protocol was optimized using C57BL/6 mice. An example of animal usage is shown for a litter of 6 pups (Figure 2). If desired, donor derived cells can be distinguished from support and recipient derived cells using congenic mouse strains. However, this is not necessary since barcoded donor cells can be distinguished based on lentiviral GFP expression.
Optional: We recommend 2 recipients for each biological replicate containing HSPCs pooled from 3 pups (Figure 2), see discussion (Notes). - Virus: Concentrated lentiviral supernatants containing barcode library
- Retronectin (Takara Bio, Clontech, catalog number: T100A )
- Sterile PBS (GE Healthcare, catalog number: SH30028.02 )
- Antibodies
Ter119 biotin (Biolegend, catalog number: 116204 )
Ter119 PE-Cy5 (Biolegend, catalog number: 116210 )
CD3 PE-Cy5 (Biolegend, catalog number: 100310 )
Gr1 PE-Cy5 (Biolegend, catalog number: 108410 )
B220 PE-Cy5 (Biolegend, catalog number: 103210 )
Sca1 PE-Cy7 (Biolegend, catalog number: 108114 )
c-kit APC (Biolegend, catalog number: 105812 ) - StemSpanTM SFEM (SFEM) (STEMCELL Technologies, catalog number: 09650 )
- Penicillin/Streptomycin (P/S) solution (Nordic Biolabs, catalog number: SV30010 )
- Cytokines
SCF (PreproTech, catalog number: 250-03 )
FLT3 (PreproTech, catalog number: 300-19 )
IL3 (PreproTech, catalog number: 213-13 )
IL6 (PreproTech, catalog number: 216-16 )
IL7 (PreproTech, catalog number: 217-17 ) - Trypan blue (Sigma-Aldrich, catalog number: T6146 )
- Anti-Biotin MicroBeads (Miltenyi Biotec, catalog number: 130-090-485 )
- 7AAD (Sigma-Aldrich, catalog number: A9400 )
- 10% bovine serum albumin (BSA) in Iscove’s MDM (STEMCELL Technologies, catalog number: 09300 )
- Ciprofloxacin, 250 mg/tablet (Teva Pharmaceutical Industries, catalog number: 01 16 40 )
- Agencourt AMPure XP beads (Beckman Coulter, catalog number: A63880 )
- Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, catalog number: Q32854 )
- High Fidelity Taq polymerase (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11304011 )
- High Sensitivity DNA Bioanalyzer Kit (Agilent Technologies, catalog number: 5067-4626 )
- BSA ≥ 98% (Sigma-Aldrich, catalog number: A9647 )
- EDTA (Sigma-Aldrich, catalog number: E5134 )
- HBSS without MgCl2 (Thermo Fisher Scientific, GibcoTM, catalog number: 14175053 )
- Sodium chloride (NaCl)
- Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 74255 )
- Proteinase K
- Staining buffer (see Recipes)
- Lysis buffer (see Recipes)
Equipment
- Dissection tools: fine tipped tweezers and scissors for FL dissection
- Pestle and mortar
- Pipette
- Centrifuge with temperature control (for 50 and 15 ml tubes) (Sigma Laborzentrifugen, model: 3-18K , catalog number: 10290)
- Centrifuge with temperature control (for Eppendorf tubes) (Beckman Coulter, model: Microfuge® 20R centrifuge , catalog number: B31607)
- MACS columns (Miltenyi Biotec, catalog number: 130-042-401 )
- MACS separator magnet (Miltenyi Biotec, model: QuadroMACSTM separator , catalog number: 130-090-976)
- Small animal heat lamp
- Cell culture room and equipment certified for lentivirus work
- Agilent 2100 Bioanalyzer Instrument (Agilent Technologies, model: 2100 Bioanalyzer Instrument )
- Qubit Fluorometer
- DynaMag 96 well plate magnet (Thermo Fisher Scientific, catalog number: 12331D )
- BD FACS Aria III (BD, model: FACS Aria III) or comparable sorter
- Deep sequencing facility and reagents (e.g., Illumina MiSeq system [Illumina, model: MiSeq system ] with MiSeq Reagent Kit v3 150-cycle or the Ion PGM System [Thermo Fisher Scientific, model: Ion PGM System ] with Ion 314 Chip Kit v2)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Kristiansen, T. A., Doyle, A. and Yuan, J. (2017). Lentiviral Barcode Labeling and Transplantation of Fetal Liver Hematopoietic Stem and Progenitor Cells. Bio-protocol 7(8): e2242. DOI: 10.21769/BioProtoc.2242.
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分类
免疫学 > 动物模型 > 小鼠
细胞生物学 > 细胞工程 > 条形码技术
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